Combination of a) n-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]2-methylphenyl}-4- (3-pyridyl)-2-pyrimidine-amine and b) a histone deacetylase inhibitor for the treatment of leukemia

ABSTRACT

The invention pertains to a combination of a histone deacetylase inhibitor and N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine or a pharmaceutically acceptable salt thereof for simultaneous, separate or sequential use for the treatment of leukemia and especially N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-resistant leukemia.

The invention relates to a combination which comprises (a) at least onehistone deacetylase inhibitor and (b)N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-amine(designated hereinafter as Compound I) or a pharmaceutically acceptablesalt thereof, and optionally at least one pharmaceutically acceptablecarrier for simultaneous, separate or sequential use, e.g. in thetreatment of leukemia; a method of treating a warm-blooded animal,especially a human, having leukemia comprising administering to theanimal (a) at least one histone deacetylase inhibitor and (b) Compound Iin a quantity which is jointly therapeutically effective for thetreatment of a leukemia; a pharmaceutical composition comprising such acombination; the use of the combination of (a) and (b) for thepreparation of a medicament for the treatment of or the delay ofprogression of leukemia; and to a commercial package or productcomprising such a combination of (a) and (b) as a combined preparationfor simultaneous, separate or sequential use.

Chronic myelogenous leukemia (CML) represents a clonal disorder of aprimitive hematopoietic stem cell that results in the progressiveaccumulation of progenitor cells that are impaired in their capacity toundergo maturation. From a pathophysiologic standpoint, the developmentof CML represents a consequence of expression of the Bcr/Abl oncogene,which encodes a fusion protein that is found in the cells of 95% ofpatients with the disease. Constitutive activation of the Bcr/Abltyrosine kinase confers hematopoietic cells with a survival advantage,contributing to leukemic transformation. In addition to protectinghematopoietic cells from certain noxious environmental stimuli (e.g.,growth factor deprivation), expression of the Bcr/Abl kinase renderscells relatively insensitive to apoptosis induced by cytotoxic drugs.Currently, the pathways downstream of Bcl/Abl responsible for apoptosisresistance in CML cells are not known with certainty. However, multiplesignaling/survival pathways have been implicated in this phenomenon,including dysregulation of NFk-B, StatS, MEK/MAP kinase, Bcl-x_(L), andAkt, among others.

Recently, the treatment of CML has been revolutionized by theintroduction of Compound I, a orally active tyrosine kinase inhibitorthat inhibits Bcr/Abl, c-Kit, PDGF and other kinases. Compound Iinterferes with the growth of and induces apoptosis in Bcr/Abl-positiveleukemia cells in vitro. Significantly, oral administration of CompoundI to CML patients results in clinical responses in >90% patients.However, the emergence of Compound I resistance in CML patientsinitially responsive to this agent, as well as the observation thatpatients in accelerated phase CML or blast crisis are less likely torespond to Compound I, have prompted the search for additionalapproaches to the treatment of this disease.

Mechanisms of resistance to Compound I include diminished drug uptake,Bcr/Abl amplification, and mutations in the Bcr/Abl kinase domain, amongothers. One possible approach to this problem involves the combinationof Compound I with other agents that exhibit anti-leukemic activity. Inthis regard, increased activity against Bcr/Abl+ leukemic cells has beendescribed when Compound I was combined with conventional cytotoxicdrugs, arsenic trioxide, geldanamycin, or tumor necrosis factorapoptosis-inducing ligand (TRAIL). Most recently, synergisticinteractions between Compound I and pharmacologic MEK1/2 inhibitors,e.g. PD184351, U0126 or the cyclin-dependent kinase inhibitorflavopiridol in Bcr/Abl+ cells has been described, including thoseresistant to Compound I due to increased Bcr/Abl protein expression.

Histone deacetylase inhibitors (HDIs), including trichostatin A, sodiumbutyrate, suberoylanilide hydroxamic acid (SAHA), depsipeptide, MS-275,and aphicidin, among others, represent a novel class of agents that actby promoting histone acetylation, resulting in relaxation of thechromatin structure. Chromatin relaxation and uncoiling permits theexpression of diverse genes, including those involved in thedifferentiation process, e.g. p21^(CIP1). In fact, HDIs, e.g. SAHA,sodium butyrate, have been shown to induce maturation in various humanleukemia cell lines. Under some circumstances, HDIs induce apoptosisrather than maturation in human leukemia cells, although the factorsthat determine which response predominates remain obscure. HDIs alsoinduce maturation in certain Bcr/Abl+ leukemia cells, e.g. K562, aphenomenon associated with diminished activation of the MAP kinasepathway.

Compound I may modify the differentiation response of Bcr/Abl+ cells andit was surprisingly found that combining Compound I with HDIs mightpromote maturation or otherwise alter leukemic cell survival. To addressthis issue, interactions between Compound I with clinically relevantHDIs, i.e. sodium butyrate and SAHA, are examined. Co-administration ofHDIs with Compound I in several CML cell lines, e.g. K562, LAMA 84,results in disruption of multiple signaling pathways, induction ofmitochondrial injury, and a dramatic potentiation of apoptosis.Moreover, this drug combination potently induces cell death in CompoundI-resistant Bcr/Abl+ cells displaying increased Bcr/Abl expression.Together, these findings suggest that the strategy of combining CompoundI with clinically relevant HDIs warrants consideration in CML andrelated hematologic malignancies.

Reversible acetylation of histones is a major regulator of geneexpression that acts by altering accessibility of transcription factorsto DNA. In normal cells, histone deacetylase (HDA) and histoneacetyltransferase together control the level of acetylation of histonesto maintain a balance. Inhibition of HDA results in the accumulation ofhyperacetylated histones, which results in a variety of cellularresponses.

Inhibitors of HDA have been studied for their therapeutic effects oncancer cells. For example, butyric acid and its derivatives, includingsodium phenylbutyrate, have been reported to induce apoptosis in vitroin human colon carcinoma, leukemia and retinoblastoma cell lines.However, butyric acid and its derivatives are not useful pharmacologicalagents because they tend to be metabolized rapidly and have a very shorthalf-life in vivo. Other inhibitors of HDA that have been widely studiedfor their anti-cancer activities are trichostatin A and trapoxin.Trichostatin A is an antifungal and antibiotic and is a reversibleinhibitor of mammalian HDA. Trapoxin is a cyclic tetrapeptide, which isan irreversible inhibitor of mammalian HDA. Although trichostatin andtrapoxin have been studied for their anti-cancer activities, the in vivoinstability of the compounds makes them less suitable as anti-cancerdrugs. There remains a need for an active compound that is suitable fortreating tumors, including cancerous tumors, that is highly efficaciousand stable.

Surprisingly, it has now been found that the effect, in treatingleukemia, of a combination which comprises (a) at least one histonedeacetylase inhibitor and (b) Compound I or a pharmaceuticallyacceptable salt thereof is greater than the effects that can be achievedwith either type of combination partner alone, i.e. greater than theeffects of a mono-therapy using only one of the combination partners (a)and (b) as defined herein.

The present invention relates to a combination for simultaneous,separate or sequential use, such as a combined preparation or apharmaceutical fixed combination, which comprises synergisticallyeffective amounts of (a) at least one histone deacetylase inhibitor and(b) Compound I or a pharmaceutically acceptable salt thereof, whereinthe active ingredients are present in each case in free form or in theform of a pharmaceutically acceptable salt, and optionally at least onepharmaceutically acceptable carrier.

In a first embodiment, the present invention relates a method oftreating a warm-blooded animal having leukemia, comprising administeringto said animal (a) at least one histone deacetylase inhibitor and (b)Compound I in a quantity which is jointly therapeutically effectiveagainst leukemia.

The term “leukemia” as used herein includes, but is not limited to,chronic myelogenous leukemia (CML) and acute lymphocyte leukemia (ALL),especially Philadelphia-chromosome positive acute lymphocyte leukemia(Ph+ALL). Preferably, the variant of leukemia to be treated by themethods disclosed herein is CML as well as Compound I-resistantleukemia, Bcr/Abl+ leukemia resistant to Compound I.

The term “Compound I resistant leukemia” as used herein definesespecially a leukemia in which Compound I or a pharmaceuticallyacceptable salt thereof shows a reduction of its therapeuticeffectiveness, it included but is not restricted to leukemia exhibitingresistance to Compound I treatment due to Bcr/Abl gene amplification,increased expression of the Bcr/Abl protein and Abl kinase domainmutation.

The term “treatment” as used herein includes the administration of thecombination partners to a warm-blooded animal, preferably a human, inneed of such a treatment with the aim to cure the disease or to have aneffect on disease regression or on the delay of progression of thedisease.

The term “delay of progression” as used herein means that the diseaseprogression is at lest slowed down or hampered by the treatment and thatthe patient exhibit survival rate that are improved in comparison topatients not being treated or being treated with the monotherapy.

The combination partner (a) Compound I isN-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-aminehaving the formula I

Compound I is preferably used in the present invention in the form ofits monomethanesulfonate salt. Compound I can be prepared andadministered as described in WO 99/03854, especially themonomethanesulfonate salt ofN-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine-aminecan be formulated as described in Examples 4 and 6 of WO 99/03854.Compound I can be administered as marketed under the trademark GLIVEC™or GLEEVEC™. The term “Compound I” includes all the pharmaceuticallyacceptable salt thereof and may also be used in form of an hydrate orincludes crystal forms, e.g. alpha and beta crystal form, such asdescribed in the European patent application No. 998 473 published onMay 10, 2000.

The term “histone deacetylase inhibitors” as used herein includes, butis not limited to sodium butyrate, MS-275 (formerly MS-27-275),suberoylanilide hydroxamic acid (SAHA), aphacidin, depsipeptide, FK228(formerly FR901228), Trichostatin A and the compounds disclosed in theinternational patent applications WO 01/38322 (Priority date: 23 Nov.1999) and WO 02/22577 (Priority date: 1 Sep. 2000) filed in the name ofNOVARTIS AG, which are hereby incorporated by reference. In particularCompound II of the formula II

in free form or in the form of a pharmaceutically acceptable salt,preferably in the form of its lactate saltandCompound III of the formula III

in its free form or in pharmaceutically acceptable salt thereof.

Compound II is specifically disclosed in Example P2 of the internationalpatent application WO 02/22577 published in Mar. 21, 2002, and filed inthe name of NOVARTIS AG.

Compound III is specifically disclosed in Example 200 of theinternational patent application WO 02/22577 published in Mar. 21, 2002,and filed in the name of NOVARTIS AG. Compound III is in free form or inthe form of a pharmaceutically acceptable salt.

The structure of the active agents identified by code numbers, genericor trade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications). The corresponding contentthereof is hereby incorporated by reference.

The present invention pertains to a combination, such as a combinedpreparation or a pharmaceutical composition, which comprises (a) theCompound I or a pharmaceutically acceptable salt thereof, especially inthe form of its monomesylate salt, and (b) at least one histonedeacetylase inhibitor selected from sodium butyrate, MS-275 (formerlyMS-27-275), suberoylanilide hydroxamic acid (SAHA), aphacidin,depsipeptide, FK228 (formerly FR901228), Trichostatin A, Compound II andCompound III, wherein the active ingredients are present in each case infree form or in the form of a pharmaceutically acceptable salt, andoptionally at least one pharmaceutically acceptable carrier; forsimultaneous, separate or sequential use.

The present invention pertains to a combination, such as a combinedpreparation or a pharmaceutical composition, which comprises (a) theCompound I or a pharmaceutically acceptable salt thereof, especially inthe form of its monomesylate salt, and (b) at least one histonedeacetylase inhibitor selected from sodium butyrate, MS-275 (formerlyMS-27-275), suberoylanilide hydroxamic acid (SAHA), aphacidin,depsipeptide, FK228 (formerly FR901228), Compound II and Compound III,wherein the active ingredients are present in each case in free form orin the form of a pharmaceutically acceptable salt, and optionally atleast one pharmaceutically acceptable carrier; for simultaneous,separate or sequential use.

When the combination partners employed in the COMBINATION OF THEINVENTION are applied in the form as marketed as single drugs, theirdosage and mode of administration can take place in accordance with theinformation provided on the package insert of the respective marketeddrug in order to result in the beneficial effect described herein, ifnot mentioned herein otherwise.

The subject-matter of the final products, the pharmaceuticalpreparations and the claims of the patent rights cited herein-above ishereby incorporated into the present application by reference. Comprisedare likewise the corresponding stereoisomers as well as thecorresponding crystal modifications, e.g. solvates and polymorphs, whichare disclosed therein. The compounds used as active ingredients in thecombinations disclosed herein can be prepared and administered asdescribed in the cited documents, respectively, if not otherwisementioned herein.

It will be understood that references to the combination partners (a)and (b) are meant to also include the pharmaceutically acceptable salts.If these combination partners (a) and (b) have, for example, at leastone basic center, they can form acid addition salts. Corresponding acidaddition salts can also be formed having, if desired, an additionallypresent basic center. The combination partners (a) and (b) having anacid group (for example COOH) can also form salts with bases. Thecombination partner (a) or (b) or a pharmaceutically acceptable saltthereof may also be used in form of a hydrate or include other solventsused for crystallization.

The term “a combined preparation”, as used herein defines especially a“kit of parts” in the sense that the combination partners (a) and (b) asdefined above can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination partners (a)and (b), i.e., simultaneously or at different time points. The parts ofthe kit of parts can then, e.g., be administered simultaneously orchronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts.

Very preferably, the time intervals are chosen such that the effect onthe treated disease in the combined use of the parts is larger than theeffect which would be obtained by use of only any one of the combinationpartners (a) and (b). The ratio of the total amounts of the combinationpartner (a) to the combination partner (b) to be administered in thecombined preparation can be varied, e.g. in order to cope with the needsof a patient sub-population to be treated or the needs of the singlepatient which different needs can be due to the particular disease, age,sex, body weight, etc. of the patients. Preferably, there is at leastone beneficial effect, e.g., a mutual enhancing of the effect of thecombination partners (a) and (b), in particular a synergism, e.g. a morethan additive effect, additional advantageous effects, less sideeffects, a combined therapeutical effect in a non-effective dosage ofone or both of the combination partners (a) and (b), and very preferablya strong synergism of the combination partners (a) and (b).

In one embodiment of the present invention, leukemia, in particularCompound I-resistant leukemia, is treated with a combination comprisingas combination partners (a) Compound I and (b) a histone deacetylaseinhibitor selected from the group consisting of sodium butyrate, MS-275(formerly MS-27-275), suberoylanilide hydroxamic acid (SAHA), aphacidin,depsipeptide, FK228 (formerly FR901228), Trichostatin A, SAHA, CompoundII and Compound III. Preferably, the histone deacetylase inhibitor isselected from sodium butyrate, SAHA, Compound II, and Compound III.

A combination which comprises (a) Compound I or a pharmaceuticallyacceptable salt thereof, especially in the form of itsmonomethanesulfonate salt, and (b) a histone deacetylase inhibitorselected from the group consisting of sodium butyrate, MS-275 (formerlyMS-27-275). suberoylanilide hydroxamic acid (SAHA), aphacidin,depsipeptide, FK(228 (formerly FR901228), Trichostatin A, preferablyselected from the group consisting of SAHA, sodium butyrate, Compound IIand Compound III, in which the active ingredients are present in eachcase in free form or in the form of a pharmaceutically acceptable saltand optionally at least one pharmaceutically acceptable carrier, will bereferred to hereinafter as a COMBINATION OF THE INVENTION.

A combination which comprises (a) Compound I or a pharmaceuticallyacceptable salt thereof, especially in the form of itsmonomethanesulfonate salt, and (b) a histone deacetylase inhibitorselected from the group consisting of sodium butyrate, MS-275 (formerlyMS-27-275), suberoylanilide hydroxamic acid (SAHA), aphacidin,depsipeptide, FK228 (formerly FR901228), preferably selected from thegroup consisting of SAHA, sodium butyrate, Compound II and Compound III,in which the active ingredients are present in each case in free form orin the form of a pharmaceutically acceptable salt and optionally atleast one pharmaceutically acceptable carrier, will be referred tohereinafter as a COMBINATION OF THE INVENTION.

The COMBINATIONS OF THE INVENTION inhibit the growth of leukemia, e.g.CML, Compound I-resistant leukemia. In one embodiment of the invention,the proliferative disease to be treated with a COMBINATION OF THEINVENTION is leukemia, especially Bcr/Abl+ leukemia and preferablyCompound I-resistant leukemia.

All the more surprising is the experimental finding that in vivo theadministration of a COMBINATION OF THE INVENTION compared to amonotherapy applying only one of the pharmaceutically active ingredientsused in the COMBINATION OF THE INVENTION results not only in a morebeneficial, especially synergistic, e.g. anti-proliferative effect, e.g.with regard to the delay of progression of a proliferative disease orwith regard to a change in tumor volume, but also in further surprisingbeneficial effects, e.g. less side-effects and a decreased mortality andmorbidity. The COMBINATIONS OF THE INVENTION are suitable in particularin the treatment of proliferative diseases refractory tochemotherapeutics knowns as anti-cancer agents as well as proliferativediseases refractory to Compound I treatment.

A further benefit is that lower doses of the active ingredients of theCOMBINATION OF THE INVENTION can be used, for example, that the dosagesneed not only often be smaller but are also applied less frequently, orcan be used in order to diminish the incidence of side-effects like,e.g., diarrhea or nausea observed with one of the combination partnersalone. This is in accordance with the desires and requirements of thepatients to be treated.

It can be shown by established test models that a COMBINATION OF THEINVENTION results in the beneficial effects described herein before. Theperson skilled in the pertinent art is fully enabled to select arelevant test model to prove such beneficial effects. Thepharmacological activity of a COMBINATION OF THE INVENTION may, forexample, be demonstrated in a clinical study or in a test procedure asessentially described hereinafter.

Suitable clinical studies are in particular randomized, double-blind,placebo-controlled, parallel studies in cancer patients with late stagedisease. Such studies are, in particular, suitable to compare theeffects of a monotherapy using the active ingredients and a therapyusing a COMBINATION OF THE INVENTION, and to prove in particular thesynergism of the active ingredients of the COMBINATIONS OF THEINVENTION. The primary endpoints in such studies can be the effect onpain scores, analgesic use, performance status, Quality of Life scoresor time to progression of the disease. The evaluation of tumors by inregular time periods, e.g. every 4, 6 or 8 weeks, is a suitable approachto determine the effect of the COMBINATION OF THE INVENTION.

It is one objective of this invention to provide a pharmaceuticalcomposition comprising a quantity, which is jointly therapeuticallyeffective against a proliferative disease comprising the COMBINATION OFTHE INVENTION. In this composition, the combination partners (a) and (b)can be administered together, one after the other or separately in onecombined unit dosage form or in two separate unit dosage forms. The unitdosage form may also be a fixed combination.

The pharmaceutical compositions according to the invention can beprepared in a manner known per se and are those suitable for enteral,such as oral or rectal, and parenteral administration to mammals(warm-blooded animals), including man, comprising a therapeuticallyeffective amount of at least one pharmacologically active combinationpartner alone or in combination with one or more pharmaceuticallyacceptable carries, especially suitable for enteral or parenteralapplication. In one embodiment of the invention, one or more of theactive ingredients are administered intravenously.

The novel pharmaceutical composition contain, for example, from about10% to about 100%, preferably from about 20% to about 60%, of the activeingredients. Pharmaceutical preparations for the combination therapy forenteral or parenteral administration are, for example, those in unitdosage forms, such as sugar-coated tablets, tablets, capsules orsuppositories, and furthermore ampoules. If not indicated otherwise,these are prepared in a manner known per se, for example by means ofconventional mixing, granulating, sugar-coating, dissolving orlyophilizing processes. It will be appreciated that the unit content ofa combination partner contained in an individual dose of each dosageform need not in itself constitute an effective amount since thenecessary effective amount can be reached by administration of aplurality of dosage units.

In particular, a therapeutically effective amount of each of thecombination partners of the COMBINATION OF THE INVENTION may beadministered simultaneously or sequentially and in any order, and thecomponents may be administered separately or as a fixed combination. Forexample, the method of delay of progression or treatment of aproliferative disease according to the invention may comprise (i)administration of the first combination partner in free orpharmaceutically acceptable salt form and (ii) administration of thesecond combination partner in free or pharmaceutically acceptable saltform, simultaneously or sequentially in any order, in jointlytherapeutically effective amounts, preferably in synergisticallyeffective amounts, e.g. in daily dosages corresponding to the amountsdescribed herein. The individual combination partners of the COMBINATIONOF THE INVENTION can be administered separately at different timesduring the course of therapy or concurrently in divided or singlecombination forms. Furthermore, the term administering also encompassesthe use of a pro-drug of a combination partner that convert in vivo tothe combination partner as such. The instant invention is therefore tobe understood as embracing all such regimes of simultaneous oralternating treatment and the term “administering” is to be interpretedaccordingly.

The effective dosage of each of the combination partners employed in theCOMBINATION OF THE INVENTION may vary depending on the particularcompound or pharmaceutical composition employed, the mode ofadministration, the condition being treated, the severity of thecondition being treated. Thus, the dosage regimen the COMBINATION OF THEINVENTION is selected in accordance with a variety of factors includingthe route of administration and the renal and hepatic function of thepatient. A physician, clinician or veterinarian of ordinary skill canreadily determine and prescribe the effective amount of the singleactive ingredients required to prevent, counter or arrest the progressof the condition. Optimal precision in achieving concentration of theactive ingredients within the range that yields efficacy withouttoxicity requires a regimen based on the kinetics of the activeingredients' availability to target sites. This involves a considerationof the distribution, equilibrium, and elimination of the activeingredients.

119.5 mg of Compound I monomethanesulfonate correspond to 100 mg ofCOMPOUND I (free base) as active substance. Depending on species, age,individual condition, mode of administration, and the clinical picturein question, effective doses of Compound I, for example daily dosescorresponding to about 50 to 1000 mg, e.g. 50 to 800 mg of the activesubstance, preferably 50 to 600 mg, e.g. 50 to 400 mg, are administeredto warm-blooded animals of about 70 kg bodyweight. For adult patientswith leukemia, a starting dose of 400 mg daily may be recommended. Forpatients with an inadequate response after an assessment of response totherapy with 400 mg daily, dose escalation can be safely considered andpatients may be treated as long as they benefit from treatment and inthe absence of limiting toxicities.

The invention relates also to a method for administering to a humansubject suffering from a leukemia COMBINATION OF THE INVENTION wherein apharmaceutically effective amount of Compound I or a pharmaceuticallyacceptable salt thereof is administered to said human subject daily fora period exceeding 3 months. The invention relates especially to suchmethod wherein a daily dose of 50 to 800 mg of the active substance,especially 50 to 600 mg, e.g. 50 to 400 mg, is administered.

When the combination partners employed in the COMBINATION OF THEINVENTION are applied in the form as marketed as single drugs, theirdosage and mode of administration can take place in accordance with theinformation provided on the packet leaflet of the respective marketeddrug in order to result in the beneficial effect described herein, ifnot mentioned herein otherwise.

The COMBINATION OF THE INVENTION can be a combined preparation or apharmaceutical composition.

Moreover, the present invention relates to a method of treating awarm-blooded animal having a leukemia comprising administering to theanimal a COMBINATION OF THE INVENTION in a quantity which is jointlytherapeutically effective against a proliferative disease and in whichthe combination partners can also be present in the form of theirpharmaceutically acceptable salts. In one embodiment of the invention,in such method the COMBINATION OF THE INVENTION is co-administered withan anti-diarrheal agent.

Furthermore, the treatment can comprise surgery, radiotherapy,cryotherapy and immunotherapy.

The invention also relates to a method of inhibiting the formation ofmetastases in a warm-blooded animal having a leukemia which comprisesadministering to the patient a pharmaceutically effective amount of theCOMBINATION OF THE INVENTION in a quantity which is jointlytherapeutically effective against said leukemia and in which thecompounds can also be present in the form of their pharmaceuticallyacceptable salts.

The present invention pertains to the use of a COMBINATION OF THEINVENTION for the treatment of leukemia, e.g. Compound I-resistantleukemia and for the preparation of a medicament for the treatment of aleukemia.

Additionally, the present invention pertains to the use of Compound I ora pharmaceutically acceptable salt thereof in combination with for thepreparation of a medicament for the treatment of aleukemia.

Moreover, the present invention provides a commercial package comprisingas active ingredients COMBINATION OF THE INVENTION, together withinstructions for simultaneous, separate or sequential use thereof in thetreatment of leukemia, e.g. CML, Compound I resistant leukemia.

The present invention preferably relates to a method of treating awarm-blooded animal having a proliferative disease, preferably BCR/ABL+human myeloid leukemia, comprising administering to said animal acombination which comprises (a) Compound I, especially in the form ofits monomethanesulfonate salt, and (b) a histone deacetylase inhibitorselected from SAHA, sodium butyrate, Compound II and Compound III, in aquantity which is jointly therapeutically effective against saidproliferative disease and in which the compounds can also be present inthe form of their pharmaceutically acceptable salts.

The present invention preferably relates to the use of a combination,which comprises (a) Compound I or a pharmaceutically acceptable saltthereof, especially in the form of its monomethanesulfonate salt, and(b) a histone deacetylase inhibitor selected from the group consistingof SAHA, sodium butyrate, Compound II and Compound III, as describedherein, for the preparation of a medicament for the treatment of aproliferative disease, preferably BCR/ABL+ human myeloid leukemia, mostpreferably a Compound I-resistant BCR/ABL+ human myeloid leukemia.

EXAMPLE 1

Materials and Methods

Cells: K562, HL60, Jurkat and U937 human leukemia cells are purchasedfrom American Type Culture Collection, Rockville, Md. LAMA 84 cells arepurchased from the German Collection of Microorganisms and Cell Cultures(Braunschweig, Germany). All cells are cultured in RPMI 1640supplemented with sodium pyruvate, MEM essential vitamins, L-glutamate,penicillin, streptomycin, and 10% heat-inactivated FCS (Hyclone, Logan,Utah). They are maintained in a 37° C., 5% CO₂, fully humidifiedincubator, passed twice weekly, and prepared for experimental procedureswhen in log-phase growth (cell density≦4×10⁵ cells/ml).

Multidrug-resistant K562R cells are derived from the parental line bysubculturing in progressively higher concentrations of doxorubicin asdescribed previously (Yanovich et al., Cancer Res 1989, 49:4499-4503.They are cultured in the absence of doxorubicin before all of theexperimental procedures. In addition, Compound I-resistant LAMA 84cells, designated LAMA 84-R, are generated by subculturing LAM 84 cellsin progressively higher concentrations of Compound I. These cells aremaintained under selection pressure in medium containing 0.5 μM ofCompound I. I.C.₅₀ Compound I values for LAMA-S-571 and LAMA-R-571 are0.3 vs 1.8 μM respectively. For studies involving the LAMA 84-R line,cells are washed free of drug and resuspended in drug-free medium 48 hbefore experimentation.

Reagents: Compound I is prepared as a 10 mM stock solution in sterileDMSO (Sigma Chemical Co., St. Louis, Mo.). Sodium butyrate and SAHA aresupplied from Calbiochem, San Diego, Calif.; BOC-fmk and IETD-fmk arepurchased from Enzyme Products, Ltd., Livermore, Calif., and formulatedin sterile DMSO before use.

Experimental Format: Logarithmically growing cells are placed in sterileplastic T-flasks (Corning, Corning, N.Y.) to which are added thedesignated drugs and the flasks replaced in the incubator for intervals.At the end of the incubation period, cells are transferred to sterilecentrifuge tubes, pelleted by centrifugation at 400×g for 10 min at roomtemperature, and prepared for analysis as described below.

Assessment of Apoptosis: After drug exposures, cytocentrifugepreparations are stained with Wright-Giemsa and viewed by lightmicroscopy to evaluate the extent of apoptosis (i.e., cell shrinkage,nuclear condensation, formation of apoptotic bodies, etc.) as describedpreviously (Yu et al., Nat. Rev. Cancer 1:294-202). For these studies,the percentage of apoptotic cells is determined by evaluating ≧500cells/condition in triplicate. To confirm the results of morphologicalanalysis, Annexin V/PI staining is used. Annexin V/PI (BD PharMingen,San Diego, Calif.) analysis of cell death is carried out as per themanufacturer's instructions. In studies involving TNF and TNF solublereceptor, both compound are combined and maintained at room temperature20 min prior to use. For these experiments 1-2×10⁵ cells per conditionare harvested. Analysis is carried out using a Becton-Dickinson FACScancytofluorometer (Mansfield, Mass.). To further confirm the morphologyresults, TUNEL staining is used. For TUNEL staining, cytocentrifugepreparations are obtained and fixed with 4% formaldehyde. The slides aretreated with acetic acid/ethanol (1:2), stained with terminaltransferase reaction mixture containing 1× terminal transferase reactionbuffer (0.25 units/μl terminal transferase, 2.5 mM CoCl₂, and 2 pmolfluorescein-12-dUTM; Boehringer Mannheim, Indianapolis, Ind.), andvisualized using fluorescence microscopy.

Determination of MMP(ΔΨ_(m)): MMP is monitored using DiOC6 [36]. Foreach condition, 4×10⁵ cells are incubated for 15 min at 37° C. in 1 mlof 40 nM DiOC6 (Calbiochem) and subsequently analyzed using a BectonDickinson FACScan cytofluorometer with excitation and emission settingsof 488 and 525 nm, respectively. Control experiments documenting theloss of ΔΨ_(m) are performed by exposing cells to 5 μM of carbamoylcyanide m-chlorophenylhydrazone (Sigma Chemical Co.; 15 min, 37° C.), anuncoupling agent that abolishes the MMP.

Preparation of S-100 Fractions and Assessment of Cytochrome C Release:U937 cells are harvested after drug treatment as described previously(Yu et al., 2001, BBRC 286:1011-18) by centrifugation at 600×g for 10min at 4° C. and washed in PBS. Cells (4×10⁶) are lysed by incubatingfor 3 min in 100 μl of lysis buffer containing 75 mM NaCl, 8 mM Na₂HPO₄,1 mM NaH₂PO₄, 1 mM EDTA, and 350 μg/ml digitonin. The lysates arecentrifuged at 12,000×g for 5 min, and the supernatant is collected andadded to an equal volume of 2× LAEMMLI buffer. The protein samples arequantified and separated by 15% SDS-PAGE.

Immunoblot Analysis: Immunoblotting is performed as described previously(Yu et al., Nat. Rev. Cancer 1:294-202). In brief, after drug treatmentcells are pelleted by centrifugation, and lysed immediately in Laemmlibuffer [1×=30 mM Tris-base (pH 6.8), 2% SDS, 2.88 mM β-mercaptoethanol,and 10% glycerol], and briefly sonicated. Homogenates are quantifiedusing Coomassie protein assay reagent (Pierce, Rockford, Ill.). Equalamounts of protein (20 μg) are boiled for 10 min, separated by SDS-PAGE(5% stacker and 10% resolving), and electroblotted to nitrocellulosemembrane. After blocking in TBS-T (0.05%) and 5% milk for 1 h at 22° C.,the blots are incubated in fresh blocking solution with an appropriatedilution of primary antibody for 4 h at 22° C. The source of antibodiesare as follows: Bcl-x_(L), rabbit polyclonal, Santa Cruz Biotechnology;XIAP, rabbit polyclonal, R&D Systems, Minneapolis, Minn.; Mcl-1, mousemonoclonal Pharmingen, San Diego, Calif.; cyclin D1, mouse monoclonal;p21, mouse monoclonal, Pharmingen; ERK 1/2, rabbit polyclonal, CellSignaling Technology, Beverly, Mass.; phospho-ERK 1/2 (thr202/tyr204),rabbit polyclonal, Cell Signaling Technology; JNK, rabbit polyclonal,Santa Cruz Biotechnology; phospho-JNK, mouse monoclonal, Santa CruzBiotechnology; phospho-p38 MAPK, rabbit polyclonal, Cell SignalingTechnology; phospho-p70S6K(421/424), rabbit polyclonal, Cell SignalingTechnology; phosphor-STAT5, Cell Signaling Technology; pRB, mousemonoclonal, Pharmingen; under-phospho-RB, mouse monoclonal, PharMingen;caspase 3, mouse monoclonal, Transduction Laboratories, Lexington, Ky.;PARP (C-2-10), mouse monoclonal, BioMol Research Laboratories, Plymouth,Mass.; cytochrome c, mouse monoclonal, Santa Cruz Biotechnology; AIF,mouse monoclonal, Santa Cruz Biotechnology; Smac, rabbit polyclonal,Upstate Biotechnology, Lake Placid, N.Y.; caspase-8, rabbit polyclonal,Pharmingen; and α-tubulin, Calbiochem. Blots are washed 3×15 min inTBS-T and then incubated with a 1:2000 dilution of horseradishperoxidase-conjugated secondary antibody (Bio-Rad Laboratories,Hercules, Calif.) for 1 h at 22° C. Blots are again washed 3×15 min inTBS-T and then developed by enhanced chemiluminescence (Pierce,Rockford, Ill.).

Differentiation Studies

Analysis of erythroid maturation of K562 cells is performed bymonitoring the production of hemoglobin as previously described (Yang etal., J. Biol. Chem. 2001, 276:25742-52). Clonogenic Survival: Effects ofdrug treatment on the clonogenic survival of leukemia cells isdetermined using a previously described clonogenic assay (Yu et al.,Mol. Pharmacol. 2001, 60: 143-54).

Transient transfections: Plasmids encoding enhanced green fluorescenceprotein under the transciptional control of the human cytomegalovirus(CMV) immediate-early promoter (pEGFP-C2), and HA-tagged activated MEK1(S218D/S222D in pUSEEamp) are obtained from Clontech Laboratories (PaloAlto, Calif.) and Upstate Biotechnology (Lake Placid, N.Y.),respectively. A 1285-bp fragment containing the MEK1 cDNA was obtainedby Apa I and partial EcoR I digestion and inserted in-frame into the(C-terminal) multiple cloning site of pEGFP-C2. The entire MEK1 cDNA inthe fusion construct is sequenced and the reading frame is confirmed.Log-phase K562 cells is transfected in electoporation hypoosmolar buffer(Eppendorf) using a BTX electromanipulator 600. 20 μg DNA and 2.0×10⁷cells are used for each condition. After 12 hour of incubation, 20% to30% of the cells displayed green fluorescence. The brightest 10% to 20%of the total cell population is isolated by fluorescence-activated cellsorting (FACS) using a Cytomation MoFLO cell sorter. The cells are thenexposed to drugs as indicated, and examined for morphologic evidence ofapoptosis as described above.

Statistical Analysis: The significance of differences betweenexperimental conditions is determined using the two-tailed Student ttest. Analysis of synergism and antagonism is performed using MedianDose Effect analysis (Chou and Talalay, 1984, Adv. Enz. Regul. 22:27-55)in conjunction with a commercially available software program (Calcysyn;Biosoft; Ferguson, Mo.) as previously described Yu et al., 2002, CancerRes. 62:188-189).

Results

To characterize interactions between Compound I and SAHA in K562 cells,dose response studies are performed. Exposure of cells for 24 hr toCompound I concentrations as high as 300 nM neglibly induced apoptosis,while 2.0 μM SAHA administered alone is also minimally-toxic. However,when cells are exposed to SAHA in combination with 100 nM Compound I, aclear increase in apoptosis is observed (i.e., ˜20%), and for Compound Iconcentrations of 250 nM, the large majority of cells (i.e., ˜75%) areapoptotic (Table 1A). TABLE 1A K562 cells are exposed for to 2.0 μM SAHAin conjunction with the indicated concentration of Compound I afterwhich apoptosis is monitored by morphological analysis of WrightGiemsa-stained specimens as described in Methods. SAHA (μM) Compound I(nM) 0 2 0 0.6 ± 0.3  3.2 ± 1.1 100 1.1 ± 0.5 20.4 ± 3.2 150 1.3 ± 0.634.5 ± 3.8 200 1.6 ± 0.6 44.6 ± 4.1 250 2.8 ± 1.2 65.3 ± 4.3 300 6.3 ±2.1 71.2 ± 4.8

Similarly, when cells are exposed for 24 hr to 250 nM Compound I incombination with increasing concentrations of SAHA, a sharp increase inapoptosis is noted at 1.0 μM SAHA, and at SAHA concentrations of 1.5 μM,the majority of cells are apoptotic (Table 1B). TABLE 1B Cells areexposed for 24 hr to 250 nM Compound I in conjunction with thedesignated concentration of SAHA, after which apoptosis is assessed asabove. Compound I (nM) SAHA (μM) 0 250 nM 0 0.6 ± 0.2  2.7 ± 1.2 1 0.8 ±0.4 21.4 ± 3.2 1.5 1.6 ± 0.7 52.3 ± 4.3 2.0 2.7 ± 0.7 64.5 ± 4.5 2.514.5 ± 1.3  68.9 ± 4.7 3.0 28.4 ± 3.2  77.8 ± 5.1For A and B, values represent the means ± S.D. for three separateexperiments.

Median Dose Effect analysis of apoptosis induction over a range of andSAHA concentrations yielded Combination Index (CI) values lower than1.0, corresponding to a synergistic interaction (Table 1C). TABLE 1CK562 cells are exposed to varying concentrations of SAHA and Compound Iat a fixed ratio (10:1) for 24 hr after which Combination Index (CI)values for apoptosis are determined in relation to the Fraction Affected(FA) using Median Dose Effect analysis. SAHA Compound I CombinationFractional (mM) (nM) Index (CI) Effect 1.0 125 0.787 0.184 1.5 187.50.762 0.406 2.0 250 0.792 0.623 2.5 312.5 0.834 0.727 3.0 375 0.7940.893CI values <1.0 correspond to a synergistic interaction. Two additionalstudies yield similar results.

There is a striking increase in apoptosis in K562 cells exposed to 250nM Compound I+2.0 μM SAHA for 24 hr as compared to K562 cells exposed todrug-free medium; SAHA 2.0 μM (24 hr); Compound I 250 nM (24 hr) shownby the photomicrographs of TUNEL-stained cells (not shown).

Time course studies of K562 cells exposed to 250 nM Compound I±2.0 μMSAHA are performed. Whereas each of these agents administeredindividually over 48 hr minimally induced apoptosis, combined treatmentresulted in an increase in apoptosis that is first observed at 12 hr,and which reached near-maximal levels by 24 hr. After 48 hr of combinedtreatment, over 90% of cells are apoptotic (Table 2A). TABLE 2A K562cells are exposed to 2.0 μM SAHA ± 250 nM Compound I for the indicatedintervals after which the percentage of apoptotic cells is determined asdescribed in Methods. Hours Control SAHA Compound I SAHA + Compound I 00.6 ± 0.2 0.6 ± 0.2 0.6 ± 0.2  0.6 ± 0.3 6 0.6 ± 0.3 0.9 ± 0.2 1.1 ± 0.5 6.7 ± 0.8 12 0.6 ± 0.2 1.7 ± 0.5 2.1 ± 0.8 13.7 ± 2.1 18 0.6 ± 0.2 2.3± 0.8 3.3 ± 1.0 35.4 ± 3.8 24 0.6 ± 0.3 3.5 ± 1.1 3.5 ± 1.2 65.5 ± 4.848 0.6 ± 0.3 14.8 ± 2.1  21.8 ± 3.4  86.3 ± 5.9

Similar results are observed when loss of mitochondrial membranepotential (ΔΨΘ_(m)) is moitored, although Compound I by itself issomewhat more toxic in this regard after 48 hr of exposure (Table 2B).TABLE 2B K562 cells are exposed to 2.0 μM SAHA ± 250 nM Compound I forthe indicated intervals after which the percentage of cells displayingloss of mitochondrial membrane potential (ΔΨ_(m)) is determined asdescribed in Methods. Hours Control SAHA Compound I SAHA + Compound I 05.3 ± 1.8  5.4 ± 1.6  5.7 ± 1.8  5.6 ± 1.7 6 5.8 ± 1.7  8.1 ± 1.9  8.8 ±1.9 16.5 ± 2.1 12 6.1 ± 1.8 10.4 ± 2.1 11.2 ± 2.1 24.6 ± 3.4 18 5.2 ±1.6 12.3 ± 2.8 11.9 ± 2.3 45.8 ± 3.8 24 6.4 ± 1.8 16.4 ± 2.9 17.8 ± 3.465.8 ± 4.5 48 5.9 ± 1.7 20.5 ± 2.8 41.3 ± 4.3 87.2 ± 5.8Together, these findings indicate that combined treatment with CompoundI and the HDI SAHA results in early induction of mitochondrial injuryand apoptosis in Bcr/Abl+ K562 cells.

To determine whether potentiation of apoptosis in K562 cells treatedwith Compound I in conjunction with an HDI would be associated with lossof leukemic cell self-renewal capacity, clonogenic assays are performed.While a 24-hr exposure to 250 nM Compound I or 2.0 μM SAHA individuallysubstantially reduced clonogenicity (i.e. to ˜25% of control values),combined treatment resulted in greater than a 2-log reduction in colonyformation (Table 2C). TABLE 2C Cells are treated with 2.0 μM SAHA ± 250nM Compound I for 24 hr, washed free of drugs, and plated in soft agaras described in Methods. At the end of a 12-day incubation period,colonies are scored and survival expressed as a percentage relative tountreated controls. Clonogenic survival (% control) SAHA 21.8 ± 3.6Compound I 22.4 ± 4.5 SAHA + Compound I 0.42 ± 0.2

Because both Compound I and HDIs such as butyrate have been shown toinduce maturation in Bcr/Abl+ cells, an attempt is made to determine ifcombined exposure to such agents would result in an increase in K562cell differentiation. To this end, hemoglobin (Hgb) production ismonitored in K562 cells treated with SAHA±Compound I. K562 cells areexposed to 2.0 μM SAHA±250 nM Compound I for the indicated intervalsafter which differentiation was monitored by quantifying intracellularlevels of Hgb as described in Methods. In each case, values representthe means±S.D. for three separate experiments. Following a 24-hrexposure, SAHA-treated cells displayed a marked (i.e. ˜50%) increase inHgb production, while Compound I is less effective in this regard.However, cells treated with both agents do not exhibit an increase inHgb levels. After 48 hr, both SAHA- and Compound I-treated cells exhibitsubstantial increases in Hgb production. However, levels of Hgb in cellsexposed to both agents, which are largely apoptotic at this time, arelower than controls(data not shown). These findings indicate thatco-treatment of Bcr/Abl+ K562 cells with Compound I and SAHA does notpromote differentiation, but instead suggests that the extensiveapoptosis that occurs under these conditions instead prevents thisprocess.

The effects of combined exposure of K562 cells to SAHA and Compound Ifor 24 hr are then examined in relation to mitochondrial injury, caspaseactivation, and expression of apoptotic regulatory proteins. K562 cellsare exposed to 2.0 μM SAHA±250 nM Compound I for 24 hr after whichWestern analysis is employed to assess release of AIF, Smac/DIABLO, andcytochrome C into S-100 cytosolic fractions, and total cellular extractsare monitored for expression of cleaved caspase 9, caspase-3, caspase-8,PARP, and Bcr/Abl. Each lane contained 25 μg of protein; blots arestripped and re-probed for tubulin to ensure equivalent loading andtransfer. Two additional studies yielded equivalent results. Whereas theeffects of Compound I (250 nM) or SAHA (2.0 μM) individually areminimal, combined exposure of cells to these agents resulted in astriking increase in release of cytochrome c, AIF, and Smac/DIABLO intothe cytosolic, S-100 cell fraction. These events are accompanied by amarked increase in caspase-9 cleavage, and degradation of caspase-3,caspase-8, and PARP. Interestingly, whereas individual treatment haslittle effect, combined exposure to Compound I and SAHA resulted in amarked decline in levels of the Bcr/Abl protein (data not shown). Thus,treatment of Bcr/Abl+ cells with a sub-toxic concentration of Compound Iin conjunction with the HDI SAHA resulted in a marked increase inrelease of pro-apoptotic mitochondrial proteins, activation of thecaspase cascade, and reduced expression of Bcr/Abl.

Interactions between SAHA and Compound I are then examined in relationto effects on various signaling, cell cycle, and apoptotic regulatoryproteins in K562 cells. Interestingly, exposure to SAHA alone (16 hr)result in a clear reduction in expression of Raf, whereas Compound I haslittle effect. K562 cells are exposed to 2.0 μM SAHA±250 nM Compound Ifor 16 hr after which Western analysis is employed to assess expressionof Raf, phosphor-MEK1/2 and -ERK1/2, total ERK1/2, phosphor-70^(S6K)(ERK-phosphorylated sites; 421/424); phospho-jNK, phosphor-p38 MAPK,phosphor-STAT 5, phospho-Akt (423), and total Akt. K562 cells aretreated as above, and monitored for expression of Bcl-x_(L), Mcl-1, andXIAP. Following treatment with Compound I±SAHA as above, expression ofp21CIP1, under-phosphorylated pRb, total pRb, and cyclin D1 are examinedby Western analysis. CF=cleavage fragment. Each lane contained 25 μg ofprotein; blots are stripped and re-probed for tubulin to ensureequivalent loading and transfer. Two additional studies yield equivalentresults (data not shown). Co-administration of Compound I and SAHAresulted in a further diminution in Raf expression. Roughly parallelchanges in levels of phospho-MEK1/2 and phospho-ERK1/2 are observed.Combined treatment with Compound I and SAHA also resulted in a markeddecrease in phosphorylation of p7056K on ERK-associated sites (421/424),as well as markedly diminished expression of phospho-STAT5, a target ofBcr/Abl. No changes in the expression of total Akt are noted, although amodest decline in phosphorylated (activated) Akt is observed in cellsexposed to both SAHA and Compound I. In addition, while Compound I andSAHA individually fail to modify expression of phospho-JNK, combinedtreatment resulted in a very dramatic increase in JNK activation. Aslight increase in phosphorylation of p38 MAPK is observed inSAHA-treated cells, but this does not change with addition of CompoundI. Combined treatment with Compound I and SAHA do not alter expressionof the anti-apoptotic proteins Bcl-x_(L) or XIAP. However, Compound Itreatment alone induces a small decrease in expression of theanti-apoptotic protein Mcl-1, as previously reported, while addition ofSAHA, which by itself exerted minimal effects, resulted in a furtherdiminution in Mcl-1 expression (data not shown).

Interactions between SAHA and Compound I are then examined in relationto expression of several cell cycle regulatory proteins. Treatment withSAHA resulted in a robust induction of p21^(CIP1), similar to effectsnoted in Bcr/Abl− leukemia cells. Unexpectedly, co-exposure to CompoundI substantially diminished induction of p21^(CIP1) by SAHA. Despite thisaction, combined exposure of cells to SAHA and Compound I resulted in amodest increase in expression of under-phosphorylated pRb accompanied bycleavage of both total and under-phosphorylated protein. Lastly, K562cells treated with both Compound I and SAHA displayed a clear reductionin levels of cyclin D₁, a phenomenon previously linked to induction ofapoptosis. Collectively, these findings indicate that co-exposure ofK562 cells to Compound I and SAHA results in perturbations in theexpression of multiple signaling, cell cycle, and apoptotic regulatoryproteins, including down-regulation of Raf, diminished activation ofMEK1/2, ERK1/2, and p70^(S6K), a striking activation of JNK, reducedexpression of Bcr/Abl, Mcl-1, p21^(CIP1), and cyclin D₁, anddephosphosphorylation/cleavage of pRb (data not shown).

To assess the role of caspases in these events, K562 cells are treatedfor 20 hr with Compound I+SAHA in the presence or absence of thepan-caspase inhibitor BOC-fmk or the caspase 8 inhibitor IETD-fmk (Table3). TABLE 3 K562 cells are exposed to 2.0 μM SAHA + 250 nM Compound Ifor 24 hr in the presence or absence 25 μM BOC-fmk or IETD- fmk, afterwhich apoptosis is monitored as above. Apoptosis (%) Control  0.6 ± 0.2SAHA + Compound I 50.1 ± 3.9 BOC + SAHA + Compound I  9.8 ± 2.4 IETD +SAHA + Compound I 41.5 ± 3.2Values represent the means ± S.D. for three separate experiments.

Cells are treated with SAHA+Compound I±BOC-fmk, after which release ofcytochrome c or Smac/DIABLO into the S-100 cytosolic fraction isassessed as above (not shown). Cells are treated with SAHA+CompoundI±BOC-fmk, after which Western analysis is used to assess expression ofprocaspase-3, Bcr/Abl, pRb, under phosphorylated pRb, Raf-1, Mcl-1,p21^(CIP1), and cyclin D₁. Each lane contained 25 μg of protein; blotsare stripped and re-probed for tubulin to ensure equivalent loading andtransfer. Two additional studies yield equivalent results (data notshown). BOC-fmk markedly inhibited apoptosis whereas IETD-fmk isminimally effective, suggesting a relatively minor role for theextrinsic pathway in Compound I/SAHA-mediated lethality in these cells.However, whereas BOC-fmk is ineffective in blocking cytochrome c releaseinto the cytosol in cells exposed to Compound I+SAHA, it largely blocksSmac/DIABLO release, indicating the latter represents a secondary,caspase-dependent event. As anticipated, BOC-fmk attenuated cleavage ofprocaspase-3 and both total and under-phosphorylated pRb. It alsopartially reverses the down-regulation of Bcr/Abl expression in CompoundI/SAHA-treated cells, suggesting a caspase-dependent component of thisphenomenon. In contrast, BOC-fmk has little effect on down regulation ofRaf, Mcl-1, p21^(CIP1), or cyclin D₁, indicating that these events arelargely independent of caspase activation. In separate studies,co-administration of BOC-fmk had no effect on the actions of SAHAadministered alone (data not shown).

To determine whether synergism between Compound I and SAHA could beextended to include other Bcr/Abl+ cells, parallel studies are conductedin LAMA 84 cells (Table 4). TABLE 4 LAMA 84 cells are exposed to 200 nMCompound I ± 1.0 μM SAHA for 24 hr, after which the percentage ofannexin V/PI+ cells (shown as gated figures) is determined by flowcytometry as described in Methods. Two additional studies yieldequivalent results. % Annexin V/PI Control 08.2 ± 1.8 SAHA 1 mM 15.2 ±2.4 Compound I 200 nM 13.4 ± 2.3 SAHA + Compound I 70.8 ± 4.8

Exposure of LAMA 84 cells to 1.0 μM SAHA or 200 nM Compound I alone for24 hr exerted minimal effects on cell death. However, when the agentsare combined, the large majority of cells (i.e., 70%) became apoptotic,reflected by annexin positivity. In contrast, no evidence of synergismis observed when SAHA was combined with several Bcr/Abl− leukemic celllines, including U937, HL-60, NB4, and Jurkat (Table 5). TABLE 5Bcr/Abl+ K562 cells, and several Bcr/Abl− leukemia cell lines, includingU937 monocytic leukemia, Jurkat lymphoblastic leukemia , and NB4 andHL-60 promyelocytic leukemia cells are exposed to SAHA ± Compound I for24 hr, after which the percentage of apoptotic cells is determined byexamining Wright Giemsa-stained cytospin slides as described in Methods.% of apoptotic cells Cell line control SAHA Compound I Compound I + SAHAK562 0.6 ± 0.3 6.6 ± 3.8 8.3 ± 3.2 74.3 ± 5.3  U937 0.8 ± 0.4 5.4 ± 1.90.9 ± 08  6.2 ± 3.0 Jurkat 0.8 ± 0.4 6.3 ± 3.2 1.1 ± 0.6 7.3 ± 2.0 NB41.2 ± 0.5 5.6 ± 2.3 1.8 ± 1.3 8.2 ± 2.4 HL60 2.1 ± 0.6 6.4 ± 1.9 3.1 ±1.3 11.5 ± 3.5 Concentrations for the individual cell lines are as follows:K562: SAHA 2.5 μM;Compound I 250 nM;U937: SAHA 1.5 μM;Compound I 250 nM;Jurkat: SAHA 0.75 μM;Compound I 250 nM;NB4: SAHA 1.5 μM;Compound I 250 nM;HL-60: SAHA 1.0 μM;Compound I 250 nM.In each case, values represent the means ± S.D. for three separateexperiments.

These findings indicate that synergistic interactions between SAHA andCompound I are restricted to human leukemic cells expressing the Bcr/Ablprotein.

Western analysis revealed that combined exposure of LAMA 84 cells toCompound I+SAHA results in a marked increase in cytosolic release ofcytochrome c, and a corresponding activation of caspases-9, -3, and -8(data not shown). Consistent with results obtained in K562 cells,treatment of LAMA 84 cells with the combination of Compound I and SAHAresults in down-regulation of Raf, p21^(CIP1), cyclin D₁, Mcl-1,phospho-STATS, enhanced under-phosphorylation and cleavage of pRb, and adramatic increase in JNK phosphorylation (data not shown).

Attempts are then made to establish whether such interactions could beextended to include HDIs other than SAHA. LAMA 84 cells are exposed to200 nM Compound I±1.0 μM SAHA for 24 hr, after which Western analysis isemployed to monitor cytochrome c release into the cytosolic S-100fraction, or expression of cleaved procaspase-9, cleaved procaspase-3,or procaspase-8 in total cell extracts (data not shown). CF=cleavagefragment. LAMA cells are treated as above, after which total cellularextracts are monitored for expression of Raf 1, phospho-JNK, p21^(CIP1),cyclin D₁, Mcl-1, and phospho-STATS. Each lane contained 25 μg ofprotein; blots are stripped and re-probed for tubulin to ensureequivalent loading and transfer. Two additional studies yield equivalentresults (data not shown).

To this end, K562 and LAMA 84 cells are exposed for 24 hr to theindicated concentrations of Compound I in the presence or absence ofsodium butyrate (SB; 1 or 2 mM), after which the extent of apoptosis isassessed the percentage of apoptotic cells is determined by examiningcytospin slides as described in Methods. Values represent the means±S.D.for three parate experiments (Table 6), co-administration of Compound Iwith SB resulted in a marked increase in apoptosis in both cell lines.Analogous to results obtained with SAHA, enhanced lethality isassociated with increased release of cytochrome c into the cytosol, andactivation of procaspases −3 and −9, down-regulation of Raf, p21^(CIP1),Mcl-1, cyclin D₁, and a marked activation of JNK (data not shown).Compound I + SB % of apoptotic cells control SB Compound I SB + CompoundI K562 0.6 ± 0.3 5.1 ± 2.2 10.3 ± 3.6 61.2 ± 6.2 LAMA 84 1.8 ± 0.5 6.4 ±2.8  9.3 ± 3.8 55.1 ± 5.4

Co-administration of Compound I with either MEK1/2 inhibitors or withthe cyclin-dependent kinase inhibitors flavopiridol results in enhancedlethality in Compound I-resistant K562 cells exhibiting increasedexpression of Bcr/Abl. Parallel studies involving the Compound I/HDIregimen are therefore carried out in K562R cells, derived from amulti-drug resistant cell line, as well as in Compound I-resistant LAMA84 cells (LAMA 84-R), which are generated by culturing cells inprogressively higher concentrations of Compound I (Table 7). TABLE 7Compound I-resistant K562 cells (K562R) and Compound I-resistant LAMA 84cells (LAMA 84R) are exposed to the indicated concentrations of CompoundI and SAHA for 24 hr, after which the percentage of apoptotic cells isdetermined by examining Wright Giemsa-stained cytospin slides asdescribed in Methods. Insets contain Western blots assaying Bcr/Ablprotein levels (along with tubulin controls) in resistant and sensitivecells. Each lane contained 25 μg of protein. Compound I + SAHA % ofapoptotic cells SAHA + control SAHA Compound I Compound I K562R 0.9 ±0.4  6.8 ± 3.5 17.6 ± 4.5 65.3 ± 5.2 LAMA 84R 2.2 ± 0.9 10.3 ± 3.4 14.8± 4.7 66.2 ± 6.1Values represent the means ± S.D. for three separate experiments.LAMA 84-R cells display approximately a 10-fold higher Compound I I.C.₅₀values than their sensitive counterparts (e.g., 2.1 vs 0.22 μM; data notshown). Western blots demonstrating the increase in Bcr/Abl proteinlevels for each cell line, are shown in the insets. It can be seen thatco-administration of Compound I (1.0 or 1.25 μM) for 48 hr, whichresults in only modest lethality in either cell line, with a minimallytoxic concentration of SAHA (i.e., 1.0 or 2.0: M)# induced cell death in the majority (e.g. ˜66%) of K562R and LAMA 84-Rcells. Exposure of sensitive K562 and LAMA 84 cells to these Compound Iconcentrations for 48 hr induced cell death in virtually 100% of cells(data not shown). Essentially identical results are obtained when cellsare exposed to Compound I in combination with SB (data not shown). Suchresults indicate that co-administration of Compound I with HDIseffectively increases cell death in # Compound I-resistant Bcr/Abl⁺cells, at least in those displaying increased expression of the Bcr/Ablprotein.

Finally, to assess the functional contribution of dysregulation of theRaf/MEK/MAP kinase axis to synergistic interactions between Compound Iand HDIs in Bcr/Abl+ cells, K562 cells are transiently transfected witha vector expressing either GFP alone or a constitutively activeMEK1/2/GFP fusion protein (Table 8). TABLE 8 Purified populations(e.g., >95%) expressing GFP are isolated using a Cytomation MoFLO cellsorter as described in Methods. Untransfected K562 cells displayed 91%viability and 0.01% GFP expression; K562 cells transfected with GFPalone; sorted cells displayed 95% viability and 95% GFP expression; K562cells transfected with GFP/constitutively active MEK1/2 fusion cDNA;sorted cells exhibited 95% viability and 96% GFP-expression. Sortedcells transfected with either GFP alone or the GFP/constitutively activeMEK1/2 fusion cDNA are cultured in drug-free medium for 5 hr, and thenexposed to 2.0 μM SAHA ± 250 nM Compound I for 24 hr, after whichapoptosis is monitored by examining Wright Giemsa-stained cytospinpreparations as described above. GFP GFP/MEK control 12.1 ± 1.8 11.5 ±1.9 SAHA 22.5 ± 2.6 19.8 ± 2.1 Compound I 23.8 ± 2.8 14.7 ± 2.2 CompoundI + SAHA 68.2 ± 4.6 38.4 ± 3.1Values represent the means ± S.D. for two separate determinations.* = significantly less than values for cells transfected with GFP alone;P < 0.05;** = P < 0.01.At the end of this period, the extent of apoptosis is monitored asdescribed above. Cells transfected with the constitutively active MEK1/2are modestly but significantly more resistant to Compound I-mediatedlethality than GFP alone controls (P < 0.05), consistent with earlierreports demonstrating potentiation of Compound I-induced apoptosis bypharmacologic MEK1/2 inhibitors. Moreover, transiently transfection ofcells# with mutant MEK1/2 very significantly protects cells from thelethality of the SAHA/Compound I regimen (P < 0.01). These findingssuggest that dysregulation of the Raf/MEK/MAP kinase cascade in K562cells exposed to Compound I in conjunction with HDIs plays a significantfunctional role in enhanced lethality.

The results of the present study indicate that co-administration of theBcr/Abl kinase inhibitor Compound I with clinically relevant HDIsresults in a dramatic increase in mitochondrial damage and apoptosis inBcr/Abl+ human leukemia cells. It has long been known that in humanleukemia cells, HDIs, presumably by promoting chromatin relaxation,permit the transcriptional activation of genes involved in thedifferentiation process. In this regard, HDIs such as SB have been shownto induce erythroid maturation in Bcr/Abl+ cell lines such as K562. Morerecently, attention has focused on the ability of HDIs, particularly thenewer generation of compounds, to trigger an apoptotic rather than amaturation program in human leukemia cells. The factors which determinewhether an HDI induces cell death versus differentiation in such cellshave not been fully elucidated, but the possibility that generation ofreactive oxygen species play a role in this process has been suggested.In any case, K562 cells, perhaps due to a generic resistance toapoptosis conferred by constitutive activation of the Bcr/Abl kinase andits downstream cytoprotective targets, are relatively insensitive toHDI-mediated cell death. There are several possible explanations for thefinding that co-administration of HDIs and Compound I results in amarked lowering the apoptotic threshold in these Bcr/Abl+ cells. Forexample, Compound I, by interfering with the anti-apoptotic actions ofone or more Bcr/Abl downstream cytoprotective targets, may potentiatethe capacity of HDIs to trigger the cell death cascade. Conversely,perturbations in various signaling and cell cycle regulatory pathwaysinduced by HDIs may, in conjunction with those triggered by Compound I,result in amplification of mitochondrial injury and apoptosis. Anadditional possibility is that Compound I, which has been reported toinduce maturation in Bcr/Abl+ cells, may, when combined with HDIs,initiate conflicting signals that result in apoptosis rather thandifferentiation. In this regard, the finding that dysregulation ofleukemic cell maturation represents a potent apoptotic stimulus is welldocumented. As these mechanisms are not mutually exclusive, thepossibility that more than one of them contributes to the markedincrease in cell death cannot be excluded.

Several lines of evidence support the notion that interruption of theRaf/MEK/MAP kinase cascade in Bcr/Abl+ cells by HDIs contributes to themarked induction of apoptosis by the Compound I/HDI regimen. Previousstudies have implicated perturbations in MEK/MAP kinase in HDI-mediateddifferentiation-induction in Bcr/Abl+ cells, although differences in thenature of such perturbations have been reported. For example, Riveroreported early activation of ERK in K562 cells exposed to sodiumbutyrat, whereas Witt et al., described a correlation betweenbutyrate-induced differentiation in K562 cells and inhibition of ERK.Such disparate results may reflect subline-specific differences or,alternatively, a biphasic temporal pattern of ERKactivation/down-regulation following butyrate exposure. In this regard,Compound I, which opposes ERK activation, at least at early intervals,has also been shown to promote K562 cell maturation. In accord withthese findings, we have also observed early inhibition of ERK activationin Compound I-treated K562 cells, although this was followed by a laterebound to basal levels of activity or above. Significantly, inhibitionof MEK/ERK activation in Compound I-treated K562 cells (i.e., bypharmacologic MEK1/2 inhibitors) represents a very potent stimulus formitochondrial damage and apoptosis [25]. Currently, however, littleinformation is available concerning effects of interruption of theMEK/MAP kinase pathway at upstream sites (e.g., at the level of Raf) inBcr/Abl+ cells. To the best of our knowledge, downregulation of Raf byHDIs in Bcr/Abl+ cells has not previously been described. Takentogether, these findings are compatible with the concepts that a)down-regulation of the Raf/MEK/ERK axis modifies the maturation programof HDI-treated cells, thereby promoting apoptosis; or b) disruption ofthe Raf/MEK/ERK cytoprotective pathway lowers the threshold forHDI-mediated cell death. In support of the latter possibility, we haverecently observed that pharmacologic MEK1/2/ERK blockade (e.g., byagents such as U0126) results in a marked potentiation of apoptosis inK562 cells exposed to HDIs (C. Yu and S. Grant, unpublished data).

Combined treatment with Compound I and FDIs also resulted in a strikingincrease in activation of the stress-related kinase JNK. Althoughexceptions exist, activation of stress-related kinases such as JNK andp38 MAPK generally favors cell death, whereas activation of MEK/MAPkinase exerts cytoprotective effects. In fact, the ratio of the netoutputs of the JNK and MAP kinase cascades has been shown to play a keyrole in survival/cell death decisions. It is therefore tempting tospeculate that the dramatic shift from MEK/MAP kinase to JNK signalingin Bcr/Abl+ cells exposed to the combination of Compound I and HDIscontributed to the marked potentiation of apoptosis.

In addition to disruption of the MEK1/2/ERK pathway, dysregulation ofthe CDKI p21^(CIP1) could also play a role in synergistic interactionsbetween Compound I and HDIs in Bcr/Abl+ cells. For example, interferencewith p21^(CIP1) induction (e.g., in cells expressing an antisenseconstruct) or in cells exposed to the CDK inhibitor flavopiridol), hasbeen shown to promote leukemic cell apoptosis following treatment withseveral differentiation-inducing agents, including PMA, bryostatin 1,and most recently, HDIs including butyrate and SAHA. The basis for thisphenomenon is not known with certainty, but may be related to theability of p21^(CIP1) to bind to and inhibit caspase-3. The observationsthat acetylation of histones by HDIs specifically activates thep21^(CIP1) promoter, and that p21^(CIP1) is regularly induced by HDIs,particularly in leukemic cells undergoing maturation, suggest thatincreased expression of this CDKI plays an important role inHDI-mediated maturation. The mechanism by which Compound I blocksp21^(CIP1) induction in HDI-treated cells is not clear, but could stemfrom disruption of the Raf/MEK/ERK axis, which is known to operateupstream of p₂₁ ^(CIP1). Alternatively, Compound I administration,particularly when combined with HDIs, could interfere with the Aktpathway, a downstream target of Bcr/Abl that has also been implicated inthe regulation of p21^(CIP1). The relative contributions, if any, ofHDI-associated down-regulation of Raf/MEK/ERK versus Compound I-mediatedinterference with p21^(CIP1) induction in synergistic interactionsbetween HDIs and Compound I in Bcr/Abl+ cells remain to be defined.

The potentiation of Compound I lethality by HDIs in Compound I-resistantK562 and LAMA 84 cells was similar if not greater than that which wehave previously observed in the case of combination regimens involvingpharmacologic MEK1/2 inhibitors or, more recently, the CDK inhibitorflavopiridol. Resistance to Compound I can potentially result frommultiple factors, including diminished cellular uptake, amplification ofbcr/abl and increased Bcr/Abl protein expression, pharmacokineticfactors, and mutations in the Bcr/Abl kinase domain. For reasons thatare unclear, increased expression of Bcr/Abl is the most commonmechanism of resistance in cultured cell lines, including those isolatedin our laboratory [25, 26]. However, in cells obtained from CML patientswho have developed in vivo resistance to Compound I, increased Bcr/Ablexpression is less frequently observed than mutations in the Bcr/Ablkinase domain. Of these, mutations at the Bcr/Abl kinase contact site(e.g., T315 and Y253) have been the most widely reported. In addition,Corbin et al., have recently employed site-directed mutagenesis toidentify other mutations in the Bcr/Abl kinase domain that reduce theinhibitory effects of Compound I, and which could potentially beclinically relevant. The ability of Compound I/HDI regimens to induceapoptosis in otherwise resistant K562 or LAMA 84 cells suggests thatthis strategy either circumvents the effects of increased Bcr/Ablexpression, or, alternatively, acts through pathways that operatedownstream or independently of Bcr/Abl. While such a strategy may beeffective in cells that display upregulation of Bcr/Abl, it remains tobe determined whether it would prove active in cells expressing Bcr/Ablmutations conferring resistance to Compound I. In this regard, theability of the Compound I/HDI regimen to trigger down-regulation ofBcr/Abl may be relevant, as single amino acid substitutions in thekinase domain may be unlikely to prevent such a process.

By inducing histone acetylation and uncoiling, HDIs promote theexpression of genes involved in the maturation process. Consequently,there has been interest in the use of HDIs to enhance thedifferentiation-inducing capacity of other agents, particularly inleukemia. For example, the ability of HDIs such as butyrate to overcomeleukemic cell resistance to all-trans retinoic acid (ATRA) has recentlybeen reported. Because Compound I is capable of inducing differentiationin Bcr/Abl+ cells, although to a limited extent, the possibility existsthat co-administration of HDIs might enhance this process. However, thedata presented here suggest that synergistic interactions betweenCompound I and HDIs primarily reflect induction of apoptosis rather thanmaturation. In view of the recent introduction of several novel HDIsinto clinical trials in humans, the concept of combining such agentswith Compound I for the treatment of patients with CML and relateddisorders may be feasible. Accordingly, further efforts to explore thisstrategy are underway.

EXAMPLE 2 EXAMPLE 3 Capsules with4-[(4-methyl-1-piperazin-1-ylmethyl)-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamidemethanesulfonate, β-crystal form

Capsules containing 119.5 mg of the compound named in the title(Compound I monomethanesulfonate) corresponding to 100 mg of COMPOUND I(free base) as active substance are prepared in the followingcomposition: Composition: SALT I 119.5 mg Avicel 200 mg PVPPXL 15 mgAerosil 2 mg Magnesium stearare 1.5 mg 338 mg

The capsules are prepared by mixing the components and filling themixture into hard gelatin capsules, size 1.

1. A combination for simultaneous, separate or sequential use whichcomprises (a)N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidineof formula I

or a pharmaceutically acceptable salt thereof and (b) at least onehistone deacetylase inhibitor in free form of in a pharmaceuticallyacceptable salt form thereof and optionally at least onepharmaceutically acceptable carrier.
 2. A combination according to claim1 wherein (b) is selected from the group consisting of sodium butyrate,MS-275, SAHA, aphacidin, depsipeptide, FK 228, trichostatin A, Compoundof formula II or a pharmaceutically acceptable salt thereof

and Compound of formula III or a pharmaceutically acceptable saltthereof,

and optionally at least one pharmaceutically acceptable carrier.
 3. Thecombination according to claim 2 wherein (b) is selected from the groupconsisting of sodium butyrate, SAHA, Compound of formula II and Compoundof formula III.
 4. Use of a combination according to claim 1 in thetreatment of a leukemia.
 5. Use of a combination according to claim 1for the preparation of a medicament in the treatment of a leukemia. 6.Use of a combination according to claim 4 wherein the leukemia is aCompound I-resistant leukemia.
 7. A method of treating a warm-bloodedanimal having a leukemia comprising administering to the animal acombination according to claim 1 in a quantity which is jointlytherapeutically effective against said leukemia and in which thecompounds can also be present in the form of their pharmaceuticallyacceptable salts.
 8. A pharmaceutical composition comprising a quantitywhich is jointly therapeutically effective against a leukemia of apharmaceutical combination according to claim 1 and at least onepharmaceutically acceptable carrier.
 9. A commercial package comprisinga combination according to claim 1 together with instructions forsimultaneous, separate or sequential use thereof in the treatment of aleukemia.
 10. (canceled)